The matrix metalloproteases (MMPs) and their endogenous regulators, the tissue inhibitors of MMPs (TIMPS) are responsible for the physiological remodeling of the extracellular matrix in healthy connective tissues. They are precisely regulated under normal physiological conditions, but when dysregulated they become a cause of many diseases such as chronic ulcers, osteoarthritis, rheumatoid arthritis, psoriasis, etc (A. L. Clutterbuck et al., Curr. Drug Targets 10(2), 1245-1254 (2009); I. Flisiak et al., Przegl Lek. 62(2), 119-122 (2005); H. Nagase et al., Cardiovascular Research 69, 562-573 (2006); X. Liu et al., Muscle Nerve 41(2), 174-178 (2010).
In view of the above, MMPs inhibitors are a potential treatment for wound healing, skin aging, psoriasis, osteoporosis, osteoarthritis, synovial inflammation, a periodontal disease and a muscle condition.
The healing process is a complex and dynamic process involving the coordinated participation of different cell types. During the first phase of healing, referred to as the inflammatory phase, platelets, neutrophils, granulocytes and macrophages play a key role in the transition between inflammation and repair by means of the release of growth factors. Growth factors released by monocytes and macrophages are necessary for starting and propagating new tissue that will cover wounds (A. J. Singer et al., N. Engl. J. Med. 341, 738-746 (1999)). During this phase, degenerated tissue is eliminated, which is a pre-requisite for optimal healing. The second phase of wound healing, referred to as the regenerative phase, is characterized by cell proliferation and extracellular matrix synthesis. In this stage of the healing process, highly vascularized tissue is formed, and several cell types, including macrophages, fibroblasts, angioblasts and myofibroblasts travel to the site of the injury. Macrophages providing a continuous source of growth factors, fibroblasts proliferate and synthesize a new extracellular matrix, which leads to the rapid formation of tissue of granulation, endothelial cells generate an angiogenesis or new vessel formation process which is stimulated by growth factors (VEGF and FGF) released by macrophages, but also by fibroblasts. In addition to angiogenic factors, the presence of a suitable extracellular matrix formed by fibronectin, and the presence of endothelial receptors which recognize this extracellular matrix are also necessary. During the final phase of the process, referred to as remodeling phase, the major cellular phenomena are the production of new connective tissue formed mainly by fibroblasts and keratinocyte proliferation and migration leading to wound re-epithelialization.
As soon as the fibroblasts synthesize collagen fibers of the new extracellular matrix, mitotic activity is reduced, as is cell density and tissue vascularization. Both collagen deposition and fibroblast orientation are determined by fibronectin, which is the most important protein of the extracellular matrix in this phase of the process (D. Greiling et al., J. Cell. Sci. 110, 861-870 (1997)). Physiological re-epithelialization is started by several stimuli: growth factors; the absence of neighboring cells at the edges of the wound, which triggers both the proliferation and migration of epidermal cells; the loss of contact of the epidermal cells with the baseline membrane, and the establishment of new interactions between cells with the components of the dermal matrix; the production and release of collagenase or MMP1 by epidermal cells and the activation of plasmin by plasminogen, which in turn activates collagenase (Fini et al., Am. J. Pathol. 149, 1287-1302 (1996)).
There are two types of skin aging, intrinsic or chronological aging and extrinsic aging, which is mostly linked to exposure to the sun (L. Rittié et al., Ageing Res. Rev. 1, 705-720 (2002)).
The intrinsic aging, also known as the natural aging process, is a continuous process that usually begins after 25 years.
For women, aging due to the reduction of estrogen production in menopause is added to the chronological process.
The firmness, elasticity and moisturizing of the skin are fundamentally consequences of the extracellular matrix of the dermis which is secreted by the cellular elements thereof, the fibroblasts, and mainly consists of type I and III collagen, responsible for its firmness and structuring, elastin, which confers the elastic properties thereof, and hyaluronic acid, the major glycosaminoglycan necessary for maintenance of moisturizing levels. Skin thickness is a consequence of both extracellular and cellular elements. The greater skin thickness, the larger the number of cells and the greater the amount of elements of the extracellular matrix there are (G. Jenkins, Mech. of Ageing Dev. 123, 801-810 (2002)).
A skin anti-aging cosmetic composition is one which is on one hand invigorating, restructuring and moisturizing, and on the other hand reduces the effects that age causes in the skin, modifying its appearance both in terms of texture and roughness.
An important anti-aging action is achieved when skin cells respond to the cosmetic composition, prolonging their life cycle, delaying the manifestation of the symptoms of cellular aging, such as the limitation of growth, the production of extracellular proteins, the increase in size or keratinization.
Another crucial property of an anti-aging product is that it should be capable of reducing the signs of age when they are already present in the skin.
Psoriasis is a disease that affects the skin. It presents a great clinical polymorphism (M A Johnson et al, Clin. Rev. Allergy. Immunol., January 27 (2012)). The skin lesion clinically manifests in the form of an erythematous plaque with well-defined edges covered by thick, whitish squamae with a waxy appearance distributed preferably by areas of extension. It is characterized by epidermal keratinocyte proliferation and failure of maturation of these cells in normal keratin formation. The evolution of the disease is unpredictable and it has been shown to greatly affect the quality of life of the patient (M M Heller et al, Dermatol. Clin. 30 (2), 281-291 (2012)).
Psoriasis is a chronic disease that has no definitive treatment. Current medical treatment depends on the type of lesion, location and age of the patient (I A Al-Hogail, Curr Vasc Pharmacol 8 (3), 432-436 (2010)).
Osteoarthritis (arthrosis), is a degenerative joint disease affecting most people after 65 years of age, and is characterized by a gradual degradation of the cartilaginous tissue, combined with the presence of inflammation and pain. Synovial inflammation usually appears later on when the disease is in its advanced stage, and it is generally only a secondary component in the pathology of osteoarthritis.
Osteoarthritis can be defined as degeneration of the hyaline articular cartilage. A secondary effect thereto is the damage of the synovial membrane and the subchondral bone (bone in contact with the cartilage), as well as new bone formation at the edges of joint surfaces.
Cartilage allows bones to move by sliding over one another. It also absorbs the tension caused by physical movement. In osteoarthritis, the surface of the cartilage breaks and wears down, causing bones to move against one another, causing friction, pain, swelling and loss of movement in the joint. The joint can become deformed over time.
Under normal conditions, cartilage renewal is a very slow process consisting of constant synthesis (anabolism) and degradation (catabolism) of the components of the extracellular matrix. Chondrocytes are the cells responsible for this metabolism, a process that must be perfectly coordinated.
Although the etiology of osteoarthritis is still unknown, it is currently accepted to be true that the first alterations occur at the chondrocyte level, which alterations will subsequently lead to the onset of an osteoarthritic joint.
A series of risk factors for the onset of the disease have been described, including: aging, genetics, obesity, overburden-induced disorders, decrease in sexual hormones, excess physical exertion in athletes, injuries or traumas, work activity and low bone mineral density.
Osteoarthritis is a disease that has no definitive treatment. There is a great need to develop disease-modifying agents to improve the quality of life of those suffering the disease, as well as to reduce the healthcare costs it entails (N. Schmitz et al., Curr. Drug Targets 11 (5), 521-527 (2010)).
Osteoporosis is a disease characterized by low bone mass and structural deterioration of bone tissue, which entails an increase in bone fragility and to a greater risk of fractures and microfractures. The most common fractures are in the hip, spinal column and wrist. It is a disease that often develops asymptomatically, so it is not detected until there is a fracture.
Bones constantly perform metabolic turnover by means of a combination of osteoblasts-induced bone formation and osteoclast-induced bone resorption. The alteration of bone metabolism can be characterized by a bone formation and bone resorption imbalance. A process of loss of bone mass begins after the age of 40 years in both sexes which is accentuated in menopausal women. With the decrease in estrogens levels, bone resorption increases and causes a negative effect on bone density.
Hip fractures are a serious result of osteoporosis. These fractures cause considerable chronic pain, disability and loss of independence, which entails a high cost for health services.
On the other hand, osteopenia is characterized by a decrease in bone mineral density which can be a precursor to osteoporosis. However, not just any person diagnosed with osteopenia will develop osteoporosis. Bone mineral density (BMD) is measured to diagnose osteopenia and osteoporosis.
Cartilage is a type of flexible connective tissue covering the joints and providing structure to the nose, ears, larynx, trachea and other parts of the body. It is a tissue without blood vessels, nerves or lymphatic vessels. Cartilaginous fish, also called elasmobranchs, such as sharks and rays, have a cartilage skeleton.
Three types of cartilaginous tissue have been described, the hyaline cartilage, fibrous cartilage and elastic cartilage. Hyaline cartilage is the most important type in the body, being found in the nose, larynx, trachea, bronchia, costal margins and articular ends of the bones.
Cartilage is formed by 70%-80% water. The main substances additionally forming cartilage are: chondrocytes, collagen, proteoglycans and hyaluronic acid. Proteoglycans mostly contain chondroitin sulfate and keratan sulfate (D. W. Fawcett, 1995, Tratado de Histologia, Ed. Interamericana McGraw-Hill, 12th Edition, Madrid; D. W. Fawcett, 1986, Textbook of Histology, Ed. Chapman and Hall, 12th Edition, New York, London; T. Aigner et al., Advanced Drug Delivery Reviews 55, 1569-1593 (2003)).
Collagen is a complex structural protein. There are several types of collagen. In cartilage, the most abundant collagen is type II. All collagens are formed by three polypeptide chains which are twisted and joined together by means of transverse bonds to form a triple helix.
Glycosaminoglycans (GAG) are high molecular weight polymeric biomolecules consisting of a repeated dimeric structure. They are fundamentally found in living organisms where they perform different physiological functions. The major glycosaminoglycan in cartilage is chondroitin sulfate which has a polymeric structure characterized by a repeating disaccharide, formed by N-acetyl-D-galactosamine and D-glucuronic acid. Most N-acetyl-D-galactosamine residues are sulfated. Chondroitin sulfate is a fundamental component of cartilage proteoglycans.
Another glycosaminoglycan that is found in cartilage is hyaluronic acid. It is a non-sulfated glycosaminoglycan with a polymeric structure characterized by a repeating disaccharide, formed by the monosaccharides N-acetyl-D-glucosamine and D-glucuronic acid.
Growth factors are substances, most being of a proteinaceous nature, which carry out an important function in intercellular communication. They are capable of stimulating cell growth and differentiation, thus regulating a wide variety of cellular processes. In the human body they carry out their function at a very low concentration, in the order of the picograms.
The use of collagen preparations for the controlled release of active substances in a wound has been described (U.S. Pat. No. 6,761,908).
EP 154447 describes a composition for wound healing consisting of an aqueous suspension of collagen and a glycosaminoglycan. While collagen-heparin and collagen-alginate compositions present good activity, the inventor stresses that collagen-chondroitin sulfate and collagen-hyaluronate preparations are less useful in the treatment of wounds.
Some methods for producing cartilage preparations are described in the literature, but they differ from the method used in the present invention and, therefore, result in cartilage products that are also different:
U.S. Pat. No. 5,503,990 describes a method for preparing finely divided bovine trachea cartilage of uniform size. This method uses enzymatic treatment to remove the unwanted protein. The cartilage that is obtained is sparingly water-soluble.
U.S. Pat. No. 3,400,199 describes a method for preparing a cartilage powder for treating wounds, with a particle size less than 40 microns and with an average particle size comprised between 5 and 10 microns. This method uses enzymatic treatment with acid-pepsin during only six hours for the purpose of removing tissue adhered to cartilage. The cartilage powder that is obtained is sparingly water-soluble.
In view of the foregoing, it is of great interest to find a method for preparing a new cartilage product containing growth factors of a natural origin at concentrations not exceeding physiological concentrations, which is water-soluble and can be useful in the treatment or prevention of wounds, signs of skin aging, psoriasis, osteoarthritis, periodontitis, muscular atrophy or osteoporosis.